Von willebrand factor-binding proteins from staphylococci

ABSTRACT

Von Willebrand factor binding proteins and polypeptides from Staphylococci are disclosed. Further, recombinant DNA molecules coding for said proteins and peptides, plasmids, phages and phagemids comprising the DNA molecules, and microorganisms and microorganisms comprising the recombinant DNA molecules or the plasmids, phages and phagemids are described. Additionally, a method of producing von Willebrand factor binding protein or polypeptide, a method of blocking the adherence of a Staphylococcus to surfaces, immobilized proteins, antigodies, immunogens, purifications methods and determination of the presence of von Willebrand factor in a complex solution, are disclosed.

[0001] The invention relates to the field of gene technology and isconcerned with recombinant DNA molecules, which contain a nucleotidesequence coding for a protein or polypeptide having vonWillebrand-binding activity. Moreover the invention comprisesmicroorganisms (including viruses) containing the aforesaid molecules,and the use thereof in the production of the aforesaid protein orpolypeptide and their use in biotechnology.

BACKGROUND OF THE INVENTION

[0002] Staphylococci

[0003] Among the coagulase positive staphylococci Staphylococcus aureusis a pathogenic species responsible for a wide variety of diseases inhumans like endocarditis, ostemyelitis, sepsis and wound infections(Espersen et al 1999). The largest populations of staphylococci arefound in regions of the skin with large numbers of sweat glands andmucous membranes surrounding openings to the body surface.

[0004] For a long time the coagulase negative staphylococci (CNS), wereconsidered as non-pathogenic, but during the last two decades they haveemerged as the most frequently isolated pathogens in nosocomialinfections. This is mainly due to an increased use of biomaterials inhuman medicine together with a larger population of immuno compromisedpatients in hospitals and an increased number of antibioticmultiresistant strains. Staphylococcus lugdunensis (Freney et al 1988)is a CNS which belong to the normal skin flora of humans butoccasionally this species can cause severe infections like endocarditis,sepicaemia and various deep tissue infections, vascular prosthesisinfection, osteomyelitis and skin infections (Espersen et at 1999,Wasserman et al 1999).

[0005] The ability of staphylococci to elicit disease in the host isgenerally due to several virulence factors like expression of adhesins,capsular polysaccharides, toxins and enzymes that can degrade hostcomponents combined with the state of the host. Binding of staphylococcito components in plasma and of the extracellular matrix (ECM) atspecific sites or structures of the host cells and tissues is thought tobe one of the major steps in the initiation of an infection. The bindingis dependent upon specific interactions between extracellular proteinsof the pathogen and ligands of the host. The relative importance ofparticular bacterial protein-ligand interactions may vary depending ondifferent factors like the site of infection or the type or stage of thedisease. Since many extracellular proteins of pathogenic staphylococciare multifuntional in their binding properties, the role of anindividual extracellular protein cannot be judged by considering aselected single binding property. Therefore it is of importance to studythese bacterial surface proteins at the molecular level. One aim of theresearch has been to study the molecular mechanisms of the respectivebacterial/host interactions in order to develop new strategies to combatinfections caused by staphylococci. The strategy has been cloning andsequencing of the bacterial genes encoding the extracellular proteinsinteracting with host components and expression of the genes in E. colito facilitate production of the proteins for further studies. Theproduced recombinant proteins have been studied with respect to theirability to prevent bacterial infections and their possible use as newbiotechnology tools (EP 163 623, EP 294 349, EP 506 923, WO 84/03103).

[0006] von Willebrand Factor

[0007] von Willebrand factor (vWF) is a large multifunctionalglycoprotein, the mature form consisting of 2050 amino acids arranged infour different types of repeats (A through D). vWF is an essentialcomponent in the maintenance of hemostasis by supporting plateletadhesion and aggregation to exposed subendothelium in damaged bloodvessels, especially under conditions of high shear forces. vWF exists asdimers about 500 kDa in size, or multimers of different sizes up to 20000 kDa. vWF is synthesised exclusively by endothelial cells andmegakaryocytes. The endothelial cells are generating a plasma pool ofvWF with a concentration of 5-10 μg/ml as well as an intracellularlystored supply of vWF in Weibel-Palade bodies. Megakaryocytes areresponsible for vWF stored within the α-granule of platelets. Thelargest multimers of vWP, with the greatest thrombogenic potential arepresent in these different storage compartments, while circulatingmultimers generally are smaller. vWF mediates platelet adhesion throughtwo distinct platelet receptors, the glycoprotein (GP) Ib in the GPIb-V-IX complex and the GP IIb-IIIa (also called integrin αIIbβ3).Further, vWF transports and stabilises the coagulation factor VIII. vWFalso binds to the endothelial vitronectin receptor (integrin αVβ3) andto various subendothelial components, such as collagens (type I, III andVI), heparin-like glucosaminoglycans, and sulfatides Vischer and deMerloose (1999), Herrmann et al (1997), Ruggeri (1999). Reduced amountof, or malfunctional vWF leads to one of several types and subtypes ofvon Willebrand disease, which is the most common inherited bleedingdisorder (Mohlke et al 1999).

[0008] An earlier report by Hartleib et al. 2000 has claimed thatProtein A, an IgG-binding protein present on cells of S. aureus is thevWF-binding protein of this species. The present invention does notrelate to protein A.

SUMMARY OF THE INVENTION

[0009] The present invention discloses new von Willebrand factor (vWF)binding proteins called, vWb (von Willebrand factor binding protein)from S. aureus and vWbl (von Willebrand factor binding protein from S.lugdunensis) DNA molecules encoding said proteins and applications fortheir use.

[0010] The invention will be described in closer detail in thefollowing, with support of the enclosed examples and drawings.

DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1. Schematic representation of the vWb protein and alignmentof inserts from the corresponding gene vWb, isolated from differentphagemid clones obtained after panning an S. aureus phage displaylibrary against recombinant vWf. S, signal sequence (signal peptidaseclevage site is between amino acids 35 and 36 in SEQ ID NO: 3); B,vWf-binding region (amino acids 368-393 in SEQ ID NO: 4). Numbers inbrackets indicate how many times an individual clone was found among the32 clones sequenced.

[0012]FIG. 2. Binding studies with phagemid particles displaying thevWF-binding domain. The number of bound phagemid particles is determinedas cfu/μl.

[0013]FIG. 3. Inhibition study with phagemid particles displaying thevwf-binding domain. The number of bound phagemid particles wasdetermined as cfu ml⁻¹, kcfu (kilo cfu). The phagemid particles werepanned against vWf in the presence of antibodies against vWb (circles)or unspecific antibodies (squares) at different concentrations. Valuesare mean±SD from two experiments.

[0014]FIG. 4. Alignment of the 10 repeat units (R1-R10) in region R ofvWbl. Since R10 is considerably more diverged than the other repeats, itis separately aligned to more clearly demonstrate the high similaritybetween the other repeats. Amino acids perfectly conserved in allrepeats are indicated with an asterisk and well conserved amino acidsbetween the repeats are indicated with a dot. The numbers indicate theamino acid position in vWbl according to SEQ ID NO: 2

[0015]FIG. 5. Schematic presentation of vWbl and alignment of insertsfrom phagemid clones obtained after pannings against rvWf. The differentregions on vWbl are indicated as S (the signal sequence), A (the nonrepetitive region) and R (encompassing 10 repeated units). The insertsindicated below vWbl (SlvW1-SlvW7) originate from pannings wherephagemid particles were eluted by lowering the pH. The insert above vWbl(SlvW8) originates from a panning procedure where phagemid particleswere not eluted. Instead E. coli TG1 cells were added directly to thewells and were allowed to get infected. The numbers indicate thepositions of amino acids in vWbl as defined in SEQ ID NO: 2.

[0016]FIG. 6. Inhibition in binding of phagemid (SlvW5) particles toimmobilised rvWf with the recombinant construct vWbl3r. Microtiter wellscoated with rvWf were separately incubated with PBS supplemented withvWbl3r or HSA or only with PBS for 1 h. One tenth of the volume (50 μl)was replaced by diluted (50×) phagestock of SlvW5. After incubation for1 h, the microtiter plates were washed with PBST and subsequently boundphagemid particles were eluted by lowering the pH to 2.1. Aliquots wereused to infect E. coli cells and plated on LAA plates. The result isshown as CFU/ml eluate. Each value is the mean of totally fourinfections from two separate wells and standard deviations areindicated.

SEQUENCE LISTING

[0017] SEQ ID NO: 1. Complete nucleotide sequence of the vwbl gene fromS. lugdunensis

[0018] SEQ ID NO: 2. The deduced amino acid sequence of the encodedprotein vWbl from S. lugdunenis.

[0019] SEQ ID NO: 3. Complete nucleotide sequence of the vwb gene fromS. aureus.

[0020] SEQ ID NO: 4. The deduced amino acid sequence of the encodedprotein vWb from S. aureus.

[0021] SEQ ID NO: 5. The mapped 24 amino acid sequence of S. lugdunensisthat binds vWF.

[0022] SEQ ID NO: 6. The mapped 26 amino acid sequence of S. aureus thatbinds vWF.

[0023] SEQ ID NO: 7-16. 67 amino acids long repeat units (R1-10) in theamino acid sequence of S. lugdunensis (SEQ ID NO: 2).

[0024] SEQ ID NO: 17. The N-terminal sequence of the purified secretedvWb protein corresponding to amino acids 36-45 in SEQ ID NO: 4.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention relates to recombinant DNA moleculescomprising nucleotide sequences, which codes for proteins orpolypeptides having vWF-binding activity. The natural sources of thesenucleotide sequences are S. aureus strain Newman and S. lugdunensisstrain 2342, respectively but with the knowledge of the nucleotide anddeduced amino acid sequences presented here, the respective gene orparts of the genes can be isolated from strains of S. aureus and S.lugdunensis, respectively or made synthetically. In particular theknowledge of the deduced amino acid sequence for the part of therespective protein responsible for the vWF-binding activity can be usedto produce syntheic polypeptides, which retain or inhibit thevWF-binding. These polypeptides can be labelled with various compoundssuch as enzymes, fluorescence, luminiscence, biotin (or derivatives of),radioactivity, etc and use e.g. in diagnostic tests such as ELISA- orRIA-techniques.

[0026] It is well known in the art that there may be few mismatches ofamino acid residues in the amino acid sequence of a protein while theprotein still retains its major characteristics. The mismatches may bereplaced of one or several amino acids, deletions of amino acid residuesor truncations of the protein. Such mismatches occur frequently ingenetic variations of native proteins. It is believed that up to 15% ofthe amino acid residues may be replaced in a protein while the proteinstill retains its major characteristics. For production of recombinantDNA molecules according to the invention a suitable cloning vehicle orvector, for example a plasmid, phagemid or phage DNA, may be cleavedwith the aid of a restriction enzyme whereupon the DNA sequence codingfor the desired protein or polypeptide is inserted into the cleavagesite to form the recombinant DNA molecule. This general procedure iswell known to a skilled person, and various techniques for cleaving andligating DNA sequences have been described in the literature (e.g. U.S.Pat. No. 4,237,224, Ausubel et al 1991, Sambrook et al 1989).Nevertheless, to the present inventors' knowledge, these techniques havenot been used for the present purpose. If the S. aureus strain Newmanand/or S. lugdunensis strain 2342, respectively are used as the sourceof the desired nucleotide sequences it is possible to isolate saidsequences and to introduce the respective sequence into a suitablevector in a manner such as described in the experimental part below or,since the nucleotide sequences are presented here, use a polymerasechain reaction (PCR)-technique to obtain the complete or fragments ofthe vwb and/or wbl genes.

[0027] Host that may be used are, microorganisms (which can be made toproduce the respective protein or active fragments thereof), which maycomprise bacterial hosts such as strains of e.g. Escherichia coli,Bacillus subtilis, Staphylococcus sp. Streptococcus sp., Lactobacilltissp. and furthermore yeasts and other eukaryotic cells in culture. Toobtain maximum expression, regulatory elements such as promoters andribosome binding sequences may be varied in a manner known per se. Theprotein or active peptide thereof can be produced intra- orextra-cellular. To obtain good secretion in various systems differentsignal peptides could be used. To facilitate purification and/ordetection the protein or fragment thereof could be fused to an affinityhandle and/or enzyme. This can be done on both genetic and proteinlevel. To modify the features of the respective protein or polypeptidethereof the gene or parts of the gene can be modified using e.g. invitro mutagenesis, or by fusion of other nucleotide sequences thatencode polypeptides resulting in a fusion protein with new features.

[0028] The invention thus comprises recombinant DNA molecules containinga nucleotide sequence, which encodes for a protein or polypeptide havingvWF-binding properties. Furthermore the invention comprises vectors suchas e.g. phagemids, plasmids and phages containing such a nucleotidesequence, and organisms, especially bacteria as e.g. strains of E. coliand Staphylococcus sp., into which such a vector has been introduced.Alternatively, such a nucleotide sequence may be integrated into thenatural genome of the microorganism.

[0029] The application furthermore relates to methods for production ofproteins or polypeptides having the vWF-binding activities of proteinvWb and Wbl, respectively or fragments thereof. According to thismethod, a microorganism as set forth above is cultured in a suitablemedium, whereupon the resultant product is isolated by some separatingmethod, for example ion exchange chromatography or by means of affinitychromatography with the aid of vWF bound to an insoluble carrier.

[0030] The invention also comprises a method to express and display anvWF-binding protein or parts thereof on a suitable virus particle e.g.bacteriophages like M13 or derivatives thereof.

[0031] Vectors, especially plasmids, which contains the respective genesvwb or wbl or parts thereof may advantageously be provided with areadily cleavable restriction site by means of which a nucleotidesequence, that codes for another product, can be fused to the respectivenucleotide sequence, in order to express a so called fusion protein. Thefusion protein may be isolated by a procedure utilising its capacity ofbinding to vWf, whereupon the other component of the system may ifdesired be liberated from the fusion protein. This technique has beendescribed at length in WO 84/03103 in respect of the protein A systemand is applicable also in the present context in an analogous manner.The fusion strategy may also be used to modify, increase or change theactivity of proteins vWb and Wbl, respectively, (or parts thereof) byfusion the proteins together or with other proteins.

[0032] The invention can also be used to affinity purify vWF. Therespective recombinant rvWF-binding protein or parts thereof can beexpressed and purified and the isolated protein or polypeptide can bebound to an insoluble carrier. The immobilized vWF-binding protein canbe used to detect and affinity purify vWF from solutions like serum. Thepresent invention also applies to the field of biotechnology thatconcerns the use of bacterial extracellular components as immunogens forvaccination against staphylococcal infections (EP 163 623, EP 294 349,EP 506 923). Immunisation using whole bacteria will always trigger ahighly polyclonal immun response with a low level of antibodies againsta given antigenic determinant. It is therefore preferable to use theprotein, polypeptide or DNA according to the present invention forimmunisation therapies. Notably, immunisation therapies can be conductedas so called passive and active immunisation. Passive immunisation usingthe invention proteins or DNA involves the raising of antibodies againstthe said protein or protein encoded by the administrated DNA in asuitable host animal, preferably a mammal, e.g. a healthy blood donor,collecting and administrating said antibodies to a patient. Another wayof generating antibodies for passive immunisation could involveproduction of specific antibodies in cell cultures. One preferredembodiment is passive immunisation of a patient prior to surgery, e.g.operations involving foreign implants in the body. Active immunisationusing the inventive protein or DNA involves the administration of thesaid protein or DNA to a patient, preferably in combination with apharmaceutically suitable immunostimulating agent. Examples of suchagents include, but are not limited to the following; cholera toxinand/or derivatives thereof, heat labile toxins, such as E. coli toxinand similar agents. The composition according to the present inventioncan further include conventional and pharmaceutical acceptable adjuvant,well known to a person skilled in the art of immunisation therapy.Preferably, in an immunisation therapy using the inventive DNA orfragments thereof, said DNA is preferably administrated intramuscularly,whereby said DNA is incorporated in suitable plasmid carriers. Anadditional gene or genes encoding a suitable immunostimulating agent canpreferably be incorporated in the same plasmid.

[0033] Said immunisation therapies are not restricted to the abovedescribed routes of administration, but can naturally be adapted to anyone of the following routes of administration: oral, nasal, subcutaneousand intramuscular.

[0034] One way of treatment of von Willebrand factor disorders is toadminister this factor to a patient using e.g. plasma or recombinanttechnology produced factor (rvWF) for review see Fischer (1999). Oneapplication of the disclosed invention is to affinity purify the vWFfrom a complex solution like serum which facilitates the purifaction ofthis factor. Furthermore the invention could also be used to determinethe concentration of vWF/rvWF in complex solutions like blood andplasma.

[0035] In particular the invention is directed to a von Willebrandfactor binding protein or polypeptide from Staphylococci, preferablyselected from the group consisting of S. aureus and S. lugdunesis.

[0036] In an an embodiment the protein or peptide has an amino acidsequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:4 and SEQ ID NOS: 5-17, and antigen determinant comprising partsthereof. The antigen determinant comprising part of one of the disclosedamino acid sequences comprises at least 5, nomally at least 7, e.g atleast 9 amino acid residues.

[0037] The invention is also directed to a recombinant DNA moleculecomprising a nucleotide sequence coding for a protein or polypeptideaccording to the invention.

[0038] In an embodiment the recombinant DNA molecule comprises at leastone nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 3, and nucleotide sequences coding for proteins andpeptides having amino acid sequences selected from SEQ ID NO: 2, SEQ IDNO: 4 and SEQ ID NOS: 5-17, and antigen determinant comprising partsthereof.

[0039] The invention is further directed to a plasmid, phage or phagemidcomprising a DNA molecule according to the invention, and to amicroorganism comprising at least one recombinant DNA molecule accordingto the invention, or at least one plasmid, phage or phagemid accordingto the invention.

[0040] An other aspect of the invention is directed to a method forproducing a von Wlllebrand factor binding protein or a polypeptidethereof, comprising the steps of

[0041] introducing at least one recombinant DNA molecule according tothe invention in a microorganism,

[0042] culturing said microorganism in a suitable medium, and

[0043] isolating the protein thus formed by chromatographicpurification.

[0044] Other aspects of the invention comprise a method for producing avon Willebrand factor binding protein or polypeptide thereof, comprisingthe step of expressing at least one recombinant protein according to theinvention on a phage particle to produce a phage particle that shows vonWillebrand factor binding activity; a method of blocking the adherenceof a Staphylococcus to surfaces, comprising addition of a proteinaccording to the invention, or an antibody according to the invention toa medium containing said Staphylococcus, preferably S. lugdunensisand/or S. aureus.

[0045] Still other aspects of the invention are directed to immobilizedproteins or peptides according to the invention. The proteins orpeptides may be coupled to glass or plastic surfaces, peptides, proteinsor carbohydrates, such as Sephadex or Dextran; and antigens specificallybinding to a protein or peptide according to the invention. Theseantibodies may be used for detection of staphylococcal infection.

[0046] Yet another aspect of the invention are directed to immunogenscomprising a protein or peptide according to the invention. These maypreferably be used in vaccines.

[0047] Further aspects of the invention comprise a method of purifyingvon Willebrand factor from a complex solution comprising chromatographywith the immobilized protein of the invention, and a method ofdetermining the presence of von Willebrand factor in a complex solutioncomprising the step of using a protein or peptide according to theinvention.

EXAMPLES

[0048] Starting Materials

[0049] Bacterial Strains, Phases and Cloning Vectors

[0050]S. aureus strains used: Newman, 8325-4, Wood 46, Ö 25, L141, U 2,12, 73. S. lugdunensis strains used: G5-87, G2-89, G16-89, G6-87,G58-88, G66-88, G3A, SÅ, 2342, 49/90, 49/91, A251 were obtained from ÅsaLjungh (Lund, Sweden). E. coli strains used: TG1, DH5-α, BL 21 (DE3),pLysS.

[0051]E. coli strain TG1 was used as bacterial host for construction ofthe library and production of the phage stocks. The E. coli phage R408(Promega, Madison, Wis., USA) was used as helper phage.

[0052] The phagemid vector pG8SAET was used to construct the phagemidlibraries (Jacobsson and Frykberg, 1999).

[0053] All strans and plasmid or phagemid constructs used in theexamples are available at the Department of Microbiology at the SwedishUniversity of Agricultural Sciences, Uppsala, Sweden.

[0054] Buffers and Media

[0055]E. coli was grown in Luria Bertani broth (LB) or on LA plates (LBcontaining 1.5% agar) (Sambrook et al 1989) at 37° C. Ampicillin was inappropriate cases added to the E. coli growth media to a final conc. of50 μg/ml. Staphylococci were grown at 37° C. on bloodagar-plates(containing 5% final conc. bovine blood) or in Tryptone Soya Broth (TSBobtained from Oxoid, Ltd Basingstoke, Hants., England) PBS: 0,05M sodiumphosphate pH 7.1, 0.9% NaCl. PBS-T: PBS supplemented with TWEEN 20 to afinal conc. of 0.05%.

[0056] Preparation of DNA from Staphylococci.

[0057] Strains of staphylococci were grown overnight in TSB. Nextmorning the cells were harvested and the chromosomal DNA prepared.

[0058] Proteins and other Reagents

[0059] Human fibrinogen was obtained from (IMCO Ltd, Stockholm, Sweden).Human serum albumin (HSA), fibronectin, human IgG and casein wereobtained from Signa, St. Louis, USA. Thrombospondin and humanvitronectin and human recombinant von Willebrand factor were obtainedfrom Åsa Ljung, Lund, Sweden. DNA probes were labelled with ³²P-ATP by arandom-priming method (Multiprime DNA labelling system; Amersham Inc,Amersham, England). Antibodies aginst human vWF was obtained fromKordia, Leiden, Netherlands. Chicken antibodies against recombinant vWbprotein were developed by Immunsystem AB, Uppsala, Sweden. Before usingthe chicken anti-vWb antibodies in various experiments they wereaffinity purified on a rvWb column. Nitrocellulose (NC)-filters (ECLfrom Amersham Pharmacia Biotech. alternatively Schleicher&Schüll,Dassel, Germany) were used to bind DNA in hybridization experiments orproteins in Western-blot techniques.

[0060] In order to analyse protein samples by native or sodium dodecylsulphate-polyacrylamid gel electrophoresis (SDS-PAGE), the PHAST-systemobtained from Pharmacia LKB Biotechnology, Uppsala, Sweden, was usedaccording to the suppliers recommendations.

[0061] Oligonucleotides used were sythesized by Life Technologies AB(Täby, Sweden). Micro Well plates (MaxiSorp, Nunc, Copenhagen, Denmark)were used in panning experiment. Plasmid DNA was prepared using QiagenMiniprep kit (Qiagen GmbH, Hilden, Germany) and the sequence of theinserts was determined as descibed by Jacobsson and Frykberg (1995,1998). The sequences obtained were analysed using the PC-gene program(Intelligenetics, Mountain View, Calif., USA). Alternatively, the NTIVector computer software (Informax Inc., North Bethesda, Md., USA) wasused for analysing the sequences obtained.

[0062] Routine Methods

[0063] Methods used routinely in molecular biology are not describedsuch as restriction of DNA with endonucleases, ligation of DNAfragments, plasmid purification etc since these methods can be found incommonly used manuals (Sambrook et al 1989, Ausubel et al 1991).Ligation reactions were performed using Ready-To-Go T4 DNA Ligase(Pharmacia, Uppsala, Sweden). The PCR reaction was performed on aMiniCycler (MJ Research Inc., Watertown, Mass., USA). DNA sequencingreactions were performed using ThermoSequenase dye terminator cyclesequencing kit (Amersham Pharmacia Biotech) and the samples wereanalysed using the using the ABI 377 DNA Sequencer (Perlin Elmer, FosterCity, Calif., USA) according to the manufacturer's instructions.

Example 1

[0064] Construction of an S. aureus Shotgun Phage Display Library.

[0065] The shotgun phage display library was constructed in principal asdescribed by Jacobson and Frykberg (1996, 1998). In short, chromosomalDNA from S. aureus strain Newman was prepared and then fragmented bysonication for different times. Sonicated DNA was analysed on an agarosegel and DNA fragments in the range of 0.5 to 5 kb were made blunt endedby treatment with T4 DNA polymerase. The DNA fragments were then ligatedinto the pG8SAET phagemid vector using the Ready-To-Go DNA ligase kit(Amersham Pharmacia Biotech). Electroporation of the ligated materialinto E. coli TG1 cells resulted in 1×10⁷ ampicillin resistanttransformants. Part of an overnight culture (4 ml) of the electroporatedbacteria was infected with helper phage R408 (10¹² plaque formingunits/ml) at a multiplicity of infection of 20 for twenty minutes andmixed with 0.5% soft agar poured onto LA plates supplemented withampicillin (LAA-plates). After incubation at 37° C. overnight, the phageparticles were released from the soft agar by vigorous shaling in LB.The suspension was centrifuged (15,000×g) for 15 minutes, followed bysterile filtration (0.45 μm). The titer of the phage display library wasdetermined to be 1.5×10⁹ colony forming units (cfu)/ml.

Example 2

[0066] Panning of the S. aureus Phase Display Library against vWF.

[0067] Microtiter wells (Maxisorp, Nunc, Copenhagen, Denmark) werecoated with 10 μl vWF (1 mg/ml) mixed with 190 μl coating buffer (0.05 MNaHCO₃, pH 9.5) and incubated at room temperature (RT), with shaking,for one hour. The wells were then washed three times with phosphatebuffered saline, 0.05% Tween 20 (PBS-T). Two hundred microliters of thephagemid library were added to the vWF coated wells, together withcasein at a final conc. of 100 μg/ml. Panning was carried out at RT,with shaking, for four hours. After washing extensively with PBS-T,bound phages were eluted with 200 μl of elution buffer (0.05 M NaCitrat,0.15 M NaCl, pH 2.0) at RT for two minutes. The eluate was neutralisedwith 25 μl of 2M Tris-buffer, pH 8.7. Different volumes (0.001 to 50 μl)of the eluate was added to 25 μl of stationary phase E. coli TG1together with LB to adjust the final volume to 200 μl. The infection wasallowed to continue for 20-30 minutes before the suspension was spreadon LAA-plates, for determining the number of infected bacteria as cfu/μlof eluate. The plates were incubated overnight at 37° C. The colonieswere counted and 150 colonies were transferred to two identical replicaplates and the rest of the colonies were collected by resuspension inLB-medium at a final volume of 0.5 ml. This suspension was infected with10 μl helper phage R408 [10¹² plaque forming units (pfu/ml)] forproduction of enriched phage stocks. The infected bacteria were mixedwith 5 ml of 0.5% soft agar, poured on a LAA-plate and incubated at 37°C. overnight. Thereafter, the soft agar were scraped off, 5 ml of LB wasadded and the mixture vortexed and vigorously shaken for three hours at37° C. The phagemids were then harvested by centrifugation (15,000×g)for 15 min. and the supernatant were sterile-filtered (0.45 μm). Thisenriched phage stock were used for subsequent repannings which werecarried out as the panning described above, but with the exception thatrepannings were performed in two hours. The enrichment of clonesexpressing the E-tag and the increase in cfu from three cycles ofpanning against vWF are shown in Table 1. TABLE 1 Number of panningcfu/μl % E-tag positive clones 1    24 8 2  50 000 70 3 182 000 94

Example 3

[0068] Screening and Sequencing of Phagemid Clones Originating from theS. aureus Phase Display Library.

[0069] After each round of panning, 150 colonies were picked inidentical pattern to two replica-plates, transferred to NC-filters(Schleicher & Schuell, Dassel, Germany) and subsequently screened forexpression of the phagemid expression tag (E-tag) with an anti-E-tagantibody (Amersham Pharmacia Biotech). Phagemid DNA from positive cloneswas prepared and the DNA sequence of the inserts were determined. Theobtained sequences were aligned and found to partially overlap eachother. Surprisingly, non of the sequenced inserts was homologous to aprevious reported S. aureus vWF-binding protein, called protein A(Hartlieb et al 2000). A schematic presentation of the overlappinginserts from different phagemid clones is shown in FIG. 1. Furthermore,the deduced amino acid sequence of the aligned inserts revealed that thebinding activity could be mapped to a 26 amino acid long sequence(TSPTTYTETTTQVPMPTVERQTQQQI, SEQ ID NO: 6, corresponding to amino acids368-393 in SEQ ID NO: 4 and nucleotides 1102-1179, in SEQ ID NO: 3). Onephagemid clone, called NvWb32 (in FIG. 1) having an insert with an openreading frame, was chosen for further studies.

Example 4

[0070] Activity of Phagemid Particles of NvWb32.

[0071] A phagemid stock of NvWb32 was prepared as follows. Five hundredmicroliters of E. coli TG1 cells harbouring the phagemid were infectedwith 10 μl helper phage R408 (10¹² pfu/ml). After propagation in softagar on an LAA plate, the phagemid particles were recovered as describedabove. The generated phage stock (2×10¹⁰ cfu/ml) was used in anexperiment to analyse the binding specificity of the phagemid particles,and it was also used in an inhibition experiment. In the bindingspecificity experiment, 200 μl of diluted phage stock (1×10⁹ cfu/ml) waspanned against untreated microtiter wells (plastic) and microtiter wellscoated with 2 μg of either fibrinogen, fibronectin, vitronectin, vonWillebrand factor, IgG, HSA or casein. After two hours of panning at RT,the wells were extensively washed with PBS-T and the bound phagemidswere eluted and allowed to infect E. coli for determination of cfu/μl ofeluate as described above. The results of this experiment are presentedin FIG. 2 which clearly shows that NvWb32 has a specificity in bindingthe vWF.

Example 5

[0072] Inhibition of NvWb32 Binding to vWF Using Antibodies againstRecombinant vWb.

[0073] The phage stock NvWb32 was diluted (5×10⁷ cfu/ml) and 90 μl wasmixed with 10 μl of various concentrations of chicken antibodies, eitherunspecific or specific against recombinant vWb (described below). Afterone hour of incubation at RT, the samples were transferred to vWF-coatedmicrotiter wells (1 μg/well) and incubated further for two hours. Thewells were extensively washed with PBS-T and the bound phagemids wereeluted and allowed to infect E. coli for determination of cfu/μl ofeluate as described above. As seen in FIG. 3 the result of thisexperiment clearly shows that antibodies raised against recombinant vWbefficiently inhibit the binding of vWb to vWF.

Example 6

[0074] Cloning of the Complete Novel Gene (vwb) Encoding the vWF-BindingProtein from S. aureus.

[0075] The genome of S. aureus is public and accessible on DNA databases like TIGR Microbial Database(http://www.tigr.org/tdb/mdb/mdbinprogress.html). To obtain the completegene (designated vwb) encoding the vWb protein the DNA inserts of theDNA sequence of the overlapping inserts presented in the example wereused to search for homologous sequences in the TIGR S. aureus genomedatabase. Computer search revealed that the overlapping inserts of theclones were contained within an open reading frame of 1551 nt (FIG. 1).Therefore, to isolate the complete vwb gene from S. aureus strain Newmantwo primers were designed: P1, primers5′-GAATTCTCATATGATTCATGAAGAAGCC-3′ (downstream) and P2,5′-GAATTCGCCATGCATTAATTATTTGCC-3′ (upstream) and used in an PCRexperiment using Pwo DNA polymerase (Roche Molecular Biochemicals,Mannheim, Germany) with chromosomal DNA from strain Newman as template.The generated PCR product was treated with T4 polynucleotide kinase togenerate blunt ends and subsequently ligated into the SmaI-site of thevector pUC18. Part of the ligation was electroporated into E. coli DH5-αfor subsequent blue-white screening. Eight white clones were isolatedand plasmids were prepared and the respective insert analysed byrestriction enzyme analysis, PCR and DNA sequencing. One clonecontaining the complete gene was further characterized. The nucleotidesequence of the complete vwb gene and the deduced amino acid sequence ofthe encoded vWb protein are presented in SEQ ID NO: 3 and SEQ ID NO: 4,respectively.

[0076] The vwb gene encodes a protein of 517 amino acids with a putativesignal sequence but without the cell wall anchoring sequence typical forsurface protein in Gram-positive bacteria. This would direct the proteinto be exported from of the bacteria and vWb can accordingly be purifiedfrom the culture supernatant.

Example 7

[0077] Using Recombinant vWb.

[0078] A part of the vwb gene was expressed in E. coli as recombinantvWb (rvWb) using the Impact T7 expression system (New England Biolabs,MA, USA) according to the manufacturer's instructions. The PCR primersP3 (downstream primer: 5′-TTAATACCATGGCTAACCCTGAATTGAAAGACTT-3′) and P4(upstream primer: 5′-ATTATTATGCGTGTGATTTGAA-3′) were used to amplify thecentral part of the vwb gene using Taq DNA polymerase from AmershamPharmacia Biotech. The PCR product was cleaved with NcoI and ligatedinto pTYB4 vector and subsequently electroporated into E. coli BL21(DE3) pLys(S). The expressed rvwb was used for generation ofantibodies in chicken and for coupling rvwb to HiTrap columns (AmershamPharmacia Biotech). Using such column, specific anti-vWb antibodies wereaffinity purified from chicken serum and used in various experiments.

Example 8

[0079] Recombinant vWb can be Used for Purification of vWF from aComplex Solution.

[0080] A HiTrap column containing immobilised rvWb was used to affinitypurify vWF from human serum. Human serum (15 ml) was passed over thecolumn (which had previously been washed with PBS) the column wasthoroughly washed with ten volumes of PBS and five volumes of PBS-T andthe bound material was eluted by lowering the pH to 3.0 using 0.1 MGlycin buffer. The eluate was TCA-percipitated as described below. Thehuman vWv was detected in western blots using anti-vWF-antibodies andsecondary HRP-labelled antibodies. Bound antibodies were detected with4-chloro-1-naphtol as substrate. The result clearly showed thatrecombinant vWb can be used to affinity purify vWF from a complexsolution such as serum.

Example 9

[0081] Purification of Wild Type vWb.

[0082]S. aureus strain Newman ΔEap, an isogenic mutant strain of S.aureus Newman in which the gene for staphylococcal extracellularadherence protein (Eap) has been deleted, was used for purification ofvWb. A culture (containing 100 ml of TSB growth medium) of S. aureusstrain ΔEap was harvested in exponential growth phase. Aftercentrifugation the supernatant was sterile filtered and subsequentlypassed through a HiTrap column with immobilised chicken anti-vWbantibodies. After washing the column with ten volumes of PBS-T and fivevolumes of PBS the bound material was eluted by lowering the pH to 3.0using 0.1 M Glycin buffer. The eluate was trichloroacetic acid(TCA)-precipitated as follows: to 1 ml of eluate, 50 μl of 100% TCA wasadded, the samples were kept on ice for 30 min. and centrifuged in amicrocentrifuge for 15 min. at 14.000 rpm at 4° C. The supernatant wasdiscarded and the pellet washed with cold acetone and again centrifugedas above. The supernatant was again discarded, the pellet was dried andresuspended in 10 μl of PBS, pH 7.4. The N-terminal sequence of thepurified secreted vWb protein was determined by Edman N-terninalsequencing. The resulting sequence obtained was VVSGEKNPYV (SEQ ID NO:17) which corresponds to amino acids 36-45 in SEQ ID NO: 2.

Example 10

[0083] SDS-PAGE and Western Blot Analysis of vWb.

[0084] Proteins samples were prepared for gel electrophoresis by mixingequal amounts of protein solution with 2×sample buffer (1×samplebuffer=62.5 mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS, 5%β-mercapto-ethanol and 0.01% bromophenol blue), boiling the mixture for5 min. and centrifuging it at 14.000 rpm for 5 min in a microcentrifuge.Supernatants were analysed by SDS-PAGE using the Phast-system (AmershamPharmacia Biotech) with PhastGel Gradient 8-25% or 4-15% gels andPhastGel SDS Buffer Strips. Proteins were blotted onto nitrocellulosefilters by diffision blot. The presence of vWb was detected either withanti-vWb antibodies and secondary RP-labelled antibodies or¹²⁵I-labelled vWF. The IODO-BEADS lodination Reagent Kit (Pierce,Rockford, Ill., USA) was used to label vWf with ¹²⁵I. Bound antibodieswere detected with 4-chloro-1-naphtol and bound ¹²⁵I-labelled vWF wasdetected with Kodak BioMax MS film (Kodak, Rochester, N.Y., USA). Theresult clearly shows that vWb can be found in the culture supernatant ofS. aureus and that vWF binds to vWb.

Example 11

[0085] The Presence of vwb in Strains of S. aureus.

[0086] Chromosomal DNA from different S. aureus strains (83254, Wood 46,Ö 25, L141, U2, 12, 73) was prepared by using the DNeasy Tissue kit fromQiagen. DNA from strain Newman and S. epidermidis strain 19 was alsoincluded in the experiment as a positive and negative control,respectively. The DNA was cleaved with EcoRI, separated on a 0.7%agarose gel and blotted to a nitrocellulose filter using the VaccuGeneblotting system (Amersham Pharmacia Biotech). After UV-fixation thefilter was probed overnight at 65° C. with a ³²P-labelled probe spanningthe complet vwb gene. After appropriate washing the filter was put on aKodak BioMax MR film for 24 hours at −70° C. before developing the film.The result showed that the vwb gene is present in all tested strains ofS. aureus.

Example 12

[0087] Construction of Shot-Gun Phase Display Library of Staphyloccuslugdunensis.

[0088] A gene library of S. lugdunensis strain 2343 was constructed inprincipal as described by Jacobson and Frykberg (1996, 1998). In short,chromosomal DNA from strain 2343 was prepared and fragmented bysonication. The sonicated DNA preparation was analysed on an agarose geland DNA fragments in the range of 0.5 to 5 kb were made blunt ended bytreatment with T4 DNA polymerase. The DNA fragments were then ligatedinto the pG8SAET phagemid vector using the Ready-To-Go DNA ligase kit(Amersham Pharmacia Biotech). Electroporation of the ligated materialinto E. coli TG1 cells resulted in 2×10⁸ ampicillin resistanttransformants. Part of an overnight culture (4 ml) of the electroporatedbacteria was infected with helper phage R408 (10¹² plaque formingunits/ml) at a multiplicity of infection of 20 for twenty minutes andmixed with 0.5% soft agar poured onto LA plates supplemented withampicillin (LAA-plates). After incubation at 37° C. overnight, the phageparticles were released from the soft agar by vigorous shaking in LB.The suspension was centrifuged (15,000×g) for 15 minutes, followed bysterile filtration (0.45 μm). The titer of the phage display library wasdetermined to be 1×10¹⁰ colony forming units (cfu)/ml.

Example 13

[0089] Panning of the S. lugdunensis Phage Display Library against vWF

[0090] A microtiter well (Maxisorp, Nunc, Copenhagen, Denmark) wascoated overnight at 4° C. with 200 μl human vWF at a conc. of 25 μg/mlin coating buffer (50 mM NaHCO₃, pH9.7). The well was washed extensivelywith PBS-T and subsequently blocked for 1 hour at RT with 200 μl ofPBS-T supplemented with 1 mg/ml casein. After washing with PBS-T, 200 μlof the phagemid library of S. lugdunensis supplemented with 0.1 mg/ml ofcasein was added and the well was incubated for 4 hours at RT. Beforeelution, the well was extensively washed with PBS-T and then eluted with200 μl buffer solution (50 mM Na-citrate; 150 mM NaCl, pH 2.0). Theeluted sample was neutralized by the addition of 25 μl 2M Trs-HCl, pH 8.Afterwards 20 μl of an E. coli TG1 overnight culture was infected with50 μl of the eluted phage particles supplemented with ˜100 μl of LBbroth. After 20 min of incubation at 37° C., the cells were spread onLAA-plates. After incubation overnight at 37° C. the colonies wereresuspended in LB broth and pooled. The pooled cells were infected withhelper phage, R408 for 20 min at RT and the sample was mixed with 5 mlof LB soft agar (0.5% agar) and poured on a LA plate. After incubationovernight the phagemid particles were extracted and subjected to anotherround of panning as previous described. The enrichment of clonesexpressing the E-tag and the increase in cfu from two cycles of panningagainst vWF are shown in Table 2. TABLE 2 Number of panning cfu/μl %E-tag positive clones 1  2 000 not tested 2 80 000 95%

Example 14

[0091] Specificity of the Phagemid Clone SlvW5 Originating from S.lugdunensis Expressing vWF Binding.

[0092] A phage stock of SlvW5 (FIG. 5) was panned against variousproteins and plastic. In the binding specificity experiment, 100 μl ofphage stock (1.3×10⁹ cfu/ml) was panned against untreated microtiterwells (plastic) and microtiter wells coated with 30 μg/ml of eitherfibrinogen, fibronectin, vitronectin, von Willebrand factor, IgG, HSA orcasein. After two hours of panning at RT, the wells were extensivelywashed with PBS-T and the bound phagemids were eluted and allowed toinfect E. coli for determination of cfu/ml of eluate as described above.The results of this experiment is presented in Table 3 which clearlyshows that SlvW5 has a specificity in binding the vWF. TABLE 3 Resultsfrom panning a phage stock (SlvW5) against immobilised ligands. Thenumber of phagemid Ligands particles per ml eluates (pH 2.1)^(a,b) rvWf3.2 × 10⁸ ± 5.9 × 10⁷ Fibrinogen 8.6 × 10⁴ ± 1.2 × 10⁴ Fibronectin 4.6 ×10⁴ ± 2.0 × 10⁴ IgG 8.0 × 10⁴ ± 2.6 × 10⁴ Vitronectin 2.4 × 10⁵ ± 3.7 ×10⁴ HSA 4.1 × 10⁵ ± 4.2 × 10⁴ Thrombospondin 2.5 × 10⁵ ± 3.5 × 10⁴ —^(c)4.0 × 10⁵ ± 3.1 × 10⁴

Example 15

[0093] Screening and Sequencing of Phagemid Clones Originating from theS. lugdunensis Phase Display Library.

[0094] A number of the vWF-binding clones (FIG. 5) were chosen forfurther studies and the DNA sequence of the inserts were determined.Sequence analysis revealed different overlapping insert coding forvWF-binding. Further analysis of the nucleotide sequences showed thatall inserts contained an open reading frame (ORF). Computer search usingthe BLAST (Basic Local Alignment Search Tool) program, where homologiesof sequences are analysed revealed that the inserts originating from S.lugdunensis were not homologous to protein A and vWb of S. aureus or toany other sequence in the data base. Furthermore, by comparing theinsert of the different clones the vWF-binding activity was mapped to asequence [FIG. 5, nt 1346-1369 in SEQ ID NO: 1] which corresponds to a24 amino acid long region [WQYTGQTTTEDGITTHIYQRIQSE, SEQ ID NO: 5].

Example 16

[0095] Cloning and Sequencing of a Gene Encoding a vWf-Binding Proteinfrom S. lugdunensis.

[0096] To isolate the complete gene encoding the putative vWf-bindingprotein, a Southern blot analysis against chromosomal DNA of strain 2342was performed. The insert of phagemid clone SlvW2 (aa 1392-1460 in SEQID NO: 2) was labelled, in a PCR procedure, and used as a probe. An ˜4kb EcoRI fragment was subsequently ligated into the corresponding siteof pUC18. Sequence analysis revealed that the chromosomal fragmentcontained the 3′-end of the gene but lacked the 5′-end. Thus, to isolatethe remaining portion of the gene, an additional Southern blotexperiment was conducted, using a probe comprising a fragment from the5′-end of the EcoRI insert. Based on the results from a Southern blotexperiment an ˜3.2 kb HincII fragment was ligated into the SmaI site ofpUC18 and subsequently the sequence of the insert was determined.Alignment of the EcoRI fragment and the HincII fragment revealed aputative ORF of 6180 nucleotides starting with a TTG codon (nucleotides22-24, SEQ ID NO:1). The ORF is preceded by a typical ribosomal bindingsequence, situated 10-17 nucleotides upstream the start codon. The gene,termed vwbl, encodes a putative protein of 2060 amino acids, SEQ ID NO:2, named vWbl (von Willebrand-binding protein of S. lugdunensis). vWblhas a putative signal sequence and the most likely site for cleavage islocated between amino acid position 47 and 48 (SEQ ID NO 2). Based onthe proposed signal sequence, the mature vWbl consists of 2013 aminoacids with a predicted molecular mass of 226 kDa. Following the signalsequence there is a region, termed A, consisting of 1255 amino acids(see FIG. 5). The A-region has no apparent similarity to other proteinsbut it harbours the interesting motif, Arg-Gly-Asp (RGD), situated atposition 1134 to 1136 in vWbl (SEQ ID NO: 2), a motif found in manyintegrin-binding proteins in mammalians as well as in cell surfaceproteins of several pathogens. The A-region is followed by a repeatregion consisting of ten units, termed R1-R10, where each unit comprises67 amino acids (SEQ ID NOS: 7-16). An alignment of the ten repeat unitsshows high similarity between them (FIG. 4). The C-terminal part of vWblharbours several characteristic features found in cell surface boundproteins of Gram-positive bacteria.

Example 17

[0097] Twenty Four Amino Acids Constitutes the “Minimal” vWf-BindingRegion in vWbl.

[0098] The vWf binding region was mapped by aligning the differentphagemid inserts from the panning experiments. This is schematicallyillustrated in FIG. 5. Despite the high similarity between most of therepeats (FIG. 4), inserts from three different panning experimentscomprised the C-terminal end of the R2 unit (SlvW1-SlvW7 in FIG. 5).Based on the alignment the “minimal” vWf-binding region in vWbl wasdetermined, from phagemid clones SlvW1 and SlvW5, to comprise 24 aminoacids ranging from position 1413 to 1436 (SEQ ID NO: 2). However, in anadditional panning experiment the panning procedure was changed. Insteadof eluting phagemid particles with low pH, E. coli TG1 cells were addeddirectly to the wells and allowed to become infected with bound phagemidparticles, followed by spreading the bacteria on LAA-plates. Thisresulted in isolation of phagemid particles comprising parts of the R5and R6 units (SlvW8 in FIG. 5) as well as clones containing the R2 unit.

Example 18

[0099] Phagemid Clone SlvW5 Binds Specifically to vWf and the Bindingcan be Inhibited by Recombinant Protein Comprising Regions R1-R3.

[0100] To investigate the binding of vWbl to vWf, a phage stock, derivedfrom SlvW5, was generated. The phage stock was separately panned againstseven host proteins and uncoated microtiter wells. The proteins used inthe assay were vWf, Fg, fibronectin, IgG, vitronectin, HSA andthrombospondin. Approximately 1000 times more phagemid particles boundto vWf than to the other proteins in the assay (Table 3). In aninhibition assay the same phage stock was used together with purifiedrecombinant protein, termed vWblr3, comprising the C-terminal end of theA-region and repeat units R1-R3 (positions 1247 to 1503 in SEQ ID NO:2). vWblr3 was incubated in vWf coated microtiter wells prior to theaddition of the phage stock. As shown in FIG. 6 the phage binding wasinhibited approximately 95% compared to the controls.

Example 19

[0101] Clinical Isolates of S. lugdunensis Possess vwbl or vwbl LikeGenes.

[0102] To investigate the distribution of the vwbl gene among clinicalisolates of S. lugdunensis chromosomal DNA was purified from strain 2342and 11 other strains (G5-87, G2-89, G16-89, G6-87, G58-88, G66-88, G3A,SÅ, 49/90, 49/91 and A251) of this species, and used in Southern blotanalysis. The DNA preparations were digested with EcoRI and probed inthe Southern blot with purified PCR product covering units R1-R10. Allstrains were found to possess a fragment that reacted with this probe.In addition, we performed PCR, with primers based on the sequence justupstream and downstream of the repeat region, in the respective DNAsamples. A fragment was amplified from ten of the twelve strains.Interestingly, the size of the PCR products varied, indicating that thenumber of repeat units in vwbl differs between S. lugdunensis strains.It was then possible to divide the ten strains into four groups,according to the sizes of the generated PCR fragments.

REFERENCES

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[0105] Fisher, E. B. (1999) Rekombinant von Willebrand Factor: PotentialTherapeutic Use. J. Thrombosis and Thrombolysis 8:197-205.

[0106] Freney, J., Brun, Y., Bes, M., Meugnier, H., Grimont, P. A. D.,Nervi, C. and Fleurette, J. (1988). Staphylococcus lugdunensis sp. nov.and Staphylococcus schleiferi sp. nov., two species from human clinicalspecimens. Int. J. Syst. Bacteriol. 38:168-172.

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[0109] Jacobsson, K. and Frykberg, L. (1996) Phage display shot-guncloning of ligand-binding domains of prokaryotic receptors approaches100% correct clones. BioTechniques 20:1070-1081.

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[0120] Patents or Patent Applications Cited:

[0121] EP 163 623

[0122] EP 294 349

[0123] EP 506 923

[0124] U.S. Pat. No. 4,237,224

[0125] WO 84/03103

1 17 1 6204 DNA Staphylococcus lugdunensis 1 caattaagga gagagaacccattgaaaagg aagaaaagca aacaaaaaga ttatttctcg 60 aatgttcaaa ataaatactccataagaaaa ttttcagtag gaattacatc aattttaata 120 ggagcatctg tcatttttggagctaattct gaagcacatg ctgcagagat aaaaagtgat 180 gataatacac aaaaagttgttaataaagaa tatagcgatg gtatttcgca agaacaacag 240 gataaatcat taaatcttgctcaagacaaa gaaaagtcaa ataatgttaa taaaaataaa 300 gtaacagacg taggaacatctgatgtgtta aaggatacaa agacagcaca tgaaaatgta 360 cagcaacaag ataatgttgcttcaaaagaa gaaactacta aaaaaacgcc tagtgctata 420 gataaaaata atacatcagcacaatctgaa gttgtaactt cgagtgatgc tactaagaca 480 tctactgctt taaatcaagttgatagaaat caactatcat cacaatctac ttcagaaaga 540 ccgaaaaaac gcgttaaacgtgacgtgggt actgatgaca agcaattagt aggggaattc 600 gtatttagcg gcacaaatagtaatgaaaga cgctataatt taacatcaga tatcgtgtct 660 aacagacgtg taacaagtacgtcattttct tattcagcat cagggcatgg cacaattgat 720 aatggcaagc tggtttacgaagcacctaaa aaatttataa tttcagaacc aacattttca 780 aaatcggaat ttgtgactaacaagacaaat ttatctgata atgagacatg gcgctatcaa 840 tttgatttaa gaccactttctggtacatca gcaggtcaaa tcaatatcag tcaagttatt 900 ggcgggatga tttggacgggaccaggtgaa ggtgatgttg ttacttcaac aatgaaaatg 960 tatcagggag atacgcttgttgatactaaa caagttaatg caacatttga tcatataaaa 1020 ggtggtttat acatagaccgagggcaagaa aaaccgacgt ctatttggag aattacacga 1080 aaatcagcgg ctgcacaatcacctacgatt ggagttatca aagaagatgg gacaatttct 1140 gagaaaacgg ataactatcgctatcaggtg ccactgccaa gagataattg gtttttagaa 1200 aatcgggatg atgcagtatcaccacattat tattcacgtt atactgattt taaatataat 1260 attttaaata ttccaagttggttagaactt gatccagatg ctaaaggcaa tagattttgg 1320 acacaagatg aatcaggcattcatctccat cttaatgaag gtgatatttt accttataat 1380 ggttatacac ccgtattaaaattgaaaaaa tctgcattaa caccagaatt attagcaaaa 1440 tttaaaactg acggctttattaaagtaaac ttagattggc aaacgatagg ccgacttcca 1500 aatggacaag attatattcaaaatgtacct gatccagagg gaattaagtt taaaatgatt 1560 aatgaggctg gtacagcaacaagtaatgtt tattttatga atttccgact gcaagagcct 1620 tcacatttgt tttcaaaagcagatcacaaa aatgaagtat tccgtgtgca aaaatatata 1680 tatgatagag accaatcgaataaatatgca cctacttaca tgcattcaat acaattacca 1740 tcagggcaag aaggcgattactttactacc tttaagcttt cattacctag aataaattat 1800 aataacggtt caagtgataaaaatgcagat aaaggaataa tattaaaagc accatttatt 1860 ttatacggtg taaatgctgatggatctact acaaaattaa acgaatggac gtcaatcaac 1920 aatacaaata atacctatggattgggagat aaaaaatata atcatttaat actccaaaca 1980 ccaattatta gagatacggtttcaccagat agtgaagcag aaaaggacat ttatggttgg 2040 gaggcagata tcacatatgcagtagatgat cagcgttggg aaacagctgc aaaagatgat 2100 gatgtgaccc aaatgcaaaatggaattatt attaatcaag tagcagaaaa tacgttacaa 2160 gctgctagtt ccgttccaacaagggcattg acaggtaatg agttacaacc agcatattcg 2220 tatattcata ctaaggatgcatttaaacac actttaacag atgttgatgt aagaaataca 2280 aattcaggtt taggaaatgtattaaactat aatgaaaaag ctaatgtcat tgtcaaagca 2340 gatactaaag attatatgaaatatttaagt acggatccat tagataatag ttctgatatt 2400 agcgttgata aattagatcatattaatcat gtctatttat cattaagtgc gcctgatgac 2460 acggcaatag gtaatgtgcgtgcatggacg aataatgaat taatacgtac atcaacagtt 2520 tggaatcctt cattatatgaaactacgcca tcgaatgttc taaaaacccg ccctgtctta 2580 aatccaatta aagtgataaaaaattataaa aatagcggta gaacgttata tatttatgaa 2640 gcacctgaag gatataagtggaatcagtcg gtaaagaatt ccattacaga acgaagttca 2700 atcactcaga atacgccagaaatcactttt gatatttata atagaggtac gttaccaacg 2760 ggaacttatt caattagatatgctacgata tgggatgaaa attcggaaat tgtgcgtcct 2820 actgaagaac aatctttaagtcataataat cttgaattat cttatgtaat tacagaagat 2880 ttaagtggta ataaaaagtttgtctcagtt attgatgtgc catttaaaat tgcattagct 2940 aaggaatatg cttctacattaactattggt aaagatgcgt cgaacagttt tgataaatct 3000 caggttgatg ttaacttaggagaaagtgtg aatttacaaa caaatacggc caactttact 3060 aacagtgaag gaattattaaagaaatcatt gtgaccattc cgaaagacaa tattaaaacg 3120 aatttaacgg cgttaattcctgacactgaa aaatatcgtg ttgtttatac aacagacacg 3180 gatgtacgta atggcgtgtacaattctaat ccaacagatt taacaaaagt tacggcagtt 3240 aaatatgttt ttgatgaaccccttgtttta acaaatggac aaagtttcca aacaaatatg 3300 cgtgttactg tgccagaggatgcacctatt ttaactaaag cgcattctca aatctttact 3360 aaaggtttgg ataatacatggctttcaggg aataaagttg agcttgaaac agaagataac 3420 cgtggagact tagtggttaagtatactaat gaatcaggga atacaattca aaattcactg 3480 acatcaaaag gtaaaaagaatacggagtat aatgttagtg tgcctcaaat gattgataga 3540 ctaaatcgac actataaatttgttagggtt gataatcaac ttgatcctac aacgggtcat 3600 tatgctaaag gtcaaactaaaattgttaat ttaatatacg tagaagtatt tgaaggtagt 3660 gtgatagccg actataaaacaacggatgga gaagtgttaa gtccgctagt aacagttgta 3720 aatagtcaaa ttgaaggaacagaatataca gctacaccag caacaattcc agatcgcgta 3780 acctttgaaa caactgatgacggtaaagtt aaaaagacaa taagttatca tttaatttcg 3840 acaccagaaa atcaatctggaacagttgta ggtaagcaga caatagaagt tcactatgta 3900 tacgaaccga ttacaacttatgaacagata ccgaacgacg cgccgcaaga aacgccagtt 3960 gcgttagaag taacacgttacgtggatagt gaaggtaatg aagtgcagga aacggaagag 4020 ggcacacatg acgcaccaggtattatcgcg gataaatggc aatatacagg ccaaacagca 4080 gcagaaaatg gtattacaacacatgtatat caacgtatcc agtcagaaat accgaatgaa 4140 gcaccacaag agacgccagtcgcgttagaa gtaacatgtt atgtggatag cgaaggtaat 4200 gaagtgcagg aaacggaagagggcacgcat gacgcaccag gtattatcgg agataaatgg 4260 caatatacag gccaaacaacaacagaagac ggtatcacaa cgcatatata tcaacgtatt 4320 caatcagaaa taccgaatgaagcaccacaa gagacgccag tcgcgttaga agtaacacgt 4380 tatgtggata gtgaaggtaatgaggtgcaa gagacagaag aaggcacgca tcaaccacca 4440 agtattatcg gagacaaatggcaatataca ggtcaaacaa caacggcaga tggcatcaca 4500 acatatgtgt atgaacgtatccagtcagaa ataccaaatg aagcgccgaa ggaaacgcca 4560 atacaattag aagtaacacgttatgtagat ggcgaaggta atgaagtgca agaaacggaa 4620 gaaggcacac atcatgcgccaggtattatc ggagacaaat ggcaatatac gggtcaaaca 4680 acaacagaat ctggcatcacgacgcatgtg tatgaacgta tacaatcgga aataccaaac 4740 gaagcaccgc aagagacaccggtacaatta gaagtaacac gttatgtgaa tagtgaaggt 4800 aatgaggtgc aagagacagaagaaggcacg catcaaccac caggtattat cggagacaaa 4860 tggcaatata caggccaaacaacaacagca gatggtatca caacatatgt gtatgaacgc 4920 attcaatcag aaataccaaatgaagcaccg aaggaaacac cggtgcaatt agaagtgaca 4980 cgctatgtgg atactgatggaaatgaagtt caagagacag aagaaggcac gcaccaaccg 5040 cctggcatta ttggagataaatggcagtat acaggaagag tcacagaaaa agatggcatc 5100 acaacgtatg tatatgaacgcatccaatca gcaatcccga acgaagcacc gcaagagaca 5160 ccggtacaat tagaagtaacgcgttatgtg gatattaccg gaaatgaagt tcaagagaca 5220 gaagaaggta cgcatcaaccgcgttatatc attggagata aatggcgtta ttctggagta 5280 acagtgacag aaaatggtattactaaacat gtctatgaac gcattcaatc aaaagttcca 5340 aatgacgcac cacaagaaacgccagtacaa ttagaagtaa cacgctatgt ggacccagaa 5400 ggaaacgaaa tacaagaaacaacagaaggt aaacatcaac cgcctggcat tattggtgac 5460 agatggcaat atacaggaaaagtcacagaa aaagatggca tcataacata tgtttatgaa 5520 cgtattcagt cagaaataccaaataatcca ccgcaagaga caccggtaga attagaagta 5580 acacgctatg tagatggcgaaggtaatgaa gtgcaagaaa caacagaagg taaacatcaa 5640 ccgcctagca ttattggagatagatggcaa tacacaggaa aagttacaga aaaagacggc 5700 attacaacat atgtctatgaacgtattcaa tcaaaagttc caaatgacgc accgcgtgta 5760 gacattgatg aattgaaaatcacaatttat gttgatacaa atggtcgtga aattgttcca 5820 tcacgaaaag gtcagttaccaccagaacaa tttatcggac aagattggca atatacagga 5880 cataagattg aaaaagatggtattacaaca tatatttata aaaaagtaga gaatgctgtg 5940 ccagcaaaac aattgaaaaagactaagcat aatacgcagt ctgaaagtca attcaaacat 6000 acaccacaag ttaaacaacaacttgttaaa tatcataatg ttaaagaaca acgttctatt 6060 gaaaagtcag aacatacagatatgcatgtg tcagagttac ctgaaacagg agaaacagct 6120 aataaaaacg gactaataggtggattgtta atagcaatag gtgcattttt cgtaacaaaa 6180 agaaaaaaag aaaacacaaaataa 6204 2 2060 PRT Staphylococcus lugdunensis 2 Leu Lys Arg Lys LysSer Lys Gln Lys Asp Tyr Phe Ser Asn Val Gln 1 5 10 15 Asn Lys Tyr SerIle Arg Lys Phe Ser Val Gly Ile Thr Ser Ile Leu 20 25 30 Ile Gly Ala SerVal Ile Phe Gly Ala Asn Ser Glu Ala His Ala Ala 35 40 45 Glu Ile Lys SerAsp Asp Asn Thr Gln Lys Val Val Asn Lys Glu Tyr 50 55 60 Ser Asp Gly IleSer Gln Glu Gln Gln Asp Lys Ser Leu Asn Leu Ala 65 70 75 80 Gln Asp LysGlu Lys Ser Asn Asn Val Asn Lys Asn Lys Val Thr Asp 85 90 95 Val Gly ThrSer Asp Val Leu Lys Asp Thr Lys Thr Ala His Glu Asn 100 105 110 Val GlnGln Gln Asp Asn Val Ala Ser Lys Glu Glu Thr Thr Lys Lys 115 120 125 ThrPro Ser Ala Ile Asp Lys Asn Asn Thr Ser Ala Gln Ser Glu Val 130 135 140Val Thr Ser Ser Asp Ala Thr Lys Thr Ser Thr Ala Leu Asn Gln Val 145 150155 160 Asp Arg Asn Gln Leu Ser Ser Gln Ser Thr Ser Glu Arg Pro Lys Lys165 170 175 Arg Val Lys Arg Asp Val Gly Thr Asp Asp Lys Gln Leu Val GlyGlu 180 185 190 Phe Val Phe Ser Gly Thr Asn Ser Asn Glu Arg Arg Tyr AsnLeu Thr 195 200 205 Ser Asp Ile Val Ser Asn Arg Arg Val Thr Ser Thr SerPhe Ser Tyr 210 215 220 Ser Ala Ser Gly His Gly Thr Ile Asp Asn Gly LysLeu Val Tyr Glu 225 230 235 240 Ala Pro Lys Lys Phe Ile Ile Ser Glu ProThr Phe Ser Lys Ser Glu 245 250 255 Phe Val Thr Asn Lys Thr Asn Leu SerAsp Asn Glu Thr Trp Arg Tyr 260 265 270 Gln Phe Asp Leu Arg Pro Leu SerGly Thr Ser Ala Gly Gln Ile Asn 275 280 285 Ile Ser Gln Val Ile Gly GlyMet Ile Trp Thr Gly Pro Gly Glu Gly 290 295 300 Asp Val Val Thr Ser ThrMet Lys Met Tyr Gln Gly Asp Thr Leu Val 305 310 315 320 Asp Thr Lys GlnVal Asn Ala Thr Phe Asp His Ile Lys Gly Gly Leu 325 330 335 Tyr Ile AspArg Gly Gln Glu Lys Pro Thr Ser Ile Trp Arg Ile Thr 340 345 350 Arg LysSer Ala Ala Ala Gln Ser Pro Thr Ile Gly Val Ile Lys Glu 355 360 365 AspGly Thr Ile Ser Glu Lys Thr Asp Asn Tyr Arg Tyr Gln Val Pro 370 375 380Leu Pro Arg Asp Asn Trp Phe Leu Glu Asn Arg Asp Asp Ala Val Ser 385 390395 400 Pro His Tyr Tyr Ser Arg Tyr Thr Asp Phe Lys Tyr Asn Ile Leu Asn405 410 415 Ile Pro Ser Trp Leu Glu Leu Asp Pro Asp Ala Lys Gly Asn ArgPhe 420 425 430 Trp Thr Gln Asp Glu Ser Gly Ile His Leu His Leu Asn GluGly Asp 435 440 445 Ile Leu Pro Tyr Asn Gly Tyr Thr Pro Val Leu Lys LeuLys Lys Ser 450 455 460 Ala Leu Thr Pro Glu Leu Leu Ala Lys Phe Lys ThrAsp Gly Phe Ile 465 470 475 480 Lys Val Asn Leu Asp Trp Gln Thr Ile GlyArg Leu Pro Asn Gly Gln 485 490 495 Asp Tyr Ile Gln Asn Val Pro Asp ProGlu Gly Ile Lys Phe Lys Met 500 505 510 Ile Asn Glu Ala Gly Thr Ala ThrSer Asn Val Tyr Phe Met Asn Phe 515 520 525 Arg Leu Gln Glu Pro Ser HisLeu Phe Ser Lys Ala Asp His Lys Asn 530 535 540 Glu Val Phe Arg Val GlnLys Tyr Ile Tyr Asp Arg Asp Gln Ser Asn 545 550 555 560 Lys Tyr Ala ProThr Tyr Met His Ser Ile Gln Leu Pro Ser Gly Gln 565 570 575 Glu Gly AspTyr Phe Thr Thr Phe Lys Leu Ser Leu Pro Arg Ile Asn 580 585 590 Tyr AsnAsn Gly Ser Ser Asp Lys Asn Ala Asp Lys Gly Ile Ile Leu 595 600 605 LysAla Pro Phe Ile Leu Tyr Gly Val Asn Ala Asp Gly Ser Thr Thr 610 615 620Lys Leu Asn Glu Trp Thr Ser Ile Asn Asn Thr Asn Asn Thr Tyr Gly 625 630635 640 Leu Gly Asp Lys Lys Tyr Asn His Leu Ile Leu Gln Thr Pro Ile Ile645 650 655 Arg Asp Thr Val Ser Pro Asp Ser Glu Ala Glu Lys Asp Ile TyrGly 660 665 670 Trp Glu Ala Asp Ile Thr Tyr Ala Val Asp Asp Gln Arg TrpGlu Thr 675 680 685 Ala Ala Lys Asp Asp Asp Val Thr Gln Met Gln Asn GlyIle Ile Ile 690 695 700 Asn Gln Val Ala Glu Asn Thr Leu Gln Ala Ala SerSer Val Pro Thr 705 710 715 720 Arg Ala Leu Thr Gly Asn Glu Leu Gln ProAla Tyr Ser Tyr Ile His 725 730 735 Thr Lys Asp Ala Phe Lys His Thr LeuThr Asp Val Asp Val Arg Asn 740 745 750 Thr Asn Ser Gly Leu Gly Asn ValLeu Asn Tyr Asn Glu Lys Ala Asn 755 760 765 Val Ile Val Lys Ala Asp ThrLys Asp Tyr Met Lys Tyr Leu Ser Thr 770 775 780 Asp Pro Leu Asp Asn SerSer Asp Ile Ser Val Asp Lys Leu Asp His 785 790 795 800 Ile Asn His ValTyr Leu Ser Leu Ser Ala Pro Asp Asp Thr Ala Ile 805 810 815 Gly Asn ValArg Ala Trp Thr Asn Asn Glu Leu Ile Arg Thr Ser Thr 820 825 830 Val TrpAsn Pro Ser Leu Tyr Glu Thr Thr Pro Ser Asn Val Leu Lys 835 840 845 ThrArg Pro Val Leu Asn Pro Ile Lys Val Ile Lys Asn Tyr Lys Asn 850 855 860Ser Gly Arg Thr Leu Tyr Ile Tyr Glu Ala Pro Glu Gly Tyr Lys Trp 865 870875 880 Asn Gln Ser Val Lys Asn Ser Ile Thr Glu Arg Ser Ser Ile Thr Gln885 890 895 Asn Thr Pro Glu Ile Thr Phe Asp Ile Tyr Asn Arg Gly Thr LeuPro 900 905 910 Thr Gly Thr Tyr Ser Ile Arg Tyr Ala Thr Ile Trp Asp GluAsn Ser 915 920 925 Glu Ile Val Arg Pro Thr Glu Glu Gln Ser Leu Ser HisAsn Asn Leu 930 935 940 Glu Leu Ser Tyr Val Ile Thr Glu Asp Leu Ser GlyAsn Lys Lys Phe 945 950 955 960 Val Ser Val Ile Asp Val Pro Phe Lys IleAla Leu Ala Lys Glu Tyr 965 970 975 Ala Ser Thr Leu Thr Ile Gly Lys AspAla Ser Asn Ser Phe Asp Lys 980 985 990 Ser Gln Val Asp Val Asn Leu GlyGlu Ser Val Asn Leu Gln Thr Asn 995 1000 1005 Thr Ala Asn Phe Thr AsnSer Glu Gly Ile Ile Lys Glu Ile Ile Val 1010 1015 1020 Thr Ile Pro LysAsp Asn Ile Lys Thr Asn Leu Thr Ala Leu Ile Pro 1025 1030 1035 1040 AspThr Glu Lys Tyr Arg Val Val Tyr Thr Thr Asp Thr Asp Val Arg 1045 10501055 Asn Gly Val Tyr Asn Ser Asn Pro Thr Asp Leu Thr Lys Val Thr Ala1060 1065 1070 Val Lys Tyr Val Phe Asp Glu Pro Leu Val Leu Thr Asn GlyGln Ser 1075 1080 1085 Phe Gln Thr Asn Met Arg Val Thr Val Pro Glu AspAla Pro Ile Leu 1090 1095 1100 Thr Lys Ala His Ser Gln Ile Phe Thr LysGly Leu Asp Asn Thr Trp 1105 1110 1115 1120 Leu Ser Gly Asn Lys Val GluLeu Glu Thr Glu Asp Asn Arg Gly Asp 1125 1130 1135 Leu Val Val Lys TyrThr Asn Glu Ser Gly Asn Thr Ile Gln Asn Ser 1140 1145 1150 Leu Thr SerLys Gly Lys Lys Asn Thr Glu Tyr Asn Val Ser Val Pro 1155 1160 1165 GlnMet Ile Asp Arg Leu Asn Arg His Tyr Lys Phe Val Arg Val Asp 1170 11751180 Asn Gln Leu Asp Pro Thr Thr Gly His Tyr Ala Lys Gly Gln Thr Lys1185 1190 1195 1200 Ile Val Asn Leu Ile Tyr Val Glu Val Phe Glu Gly SerVal Ile Ala 1205 1210 1215 Asp Tyr Lys Thr Thr Asp Gly Glu Val Leu SerPro Leu Val Thr Val 1220 1225 1230 Val Asn Ser Gln Ile Glu Gly Thr GluTyr Thr Ala Thr Pro Ala Thr 1235 1240 1245 Ile Pro Asp Arg Val Thr PheGlu Thr Thr Asp Asp Gly Lys Val Lys 1250 1255 1260 Lys Thr Ile Ser TyrHis Leu Ile Ser Thr Pro Glu Asn Gln Ser Gly 1265 1270 1275 1280 Thr ValVal Gly Lys Gln Thr Ile Glu Val His Tyr Val Tyr Glu Pro 1285 1290 1295Ile Thr Thr Tyr Glu Gln Ile Pro Asn Asp Ala Pro Gln Glu Thr Pro 13001305 1310 Val Ala Leu Glu Val Thr Arg Tyr Val Asp Ser Glu Gly Asn GluVal 1315 1320 1325 Gln Glu Thr Glu Glu Gly Thr His Asp Ala Pro Gly IleIle Ala Asp 1330 1335 1340 Lys Trp Gln Tyr Thr Gly Gln Thr Ala Ala GluAsn Gly Ile Thr Thr 1345 1350 1355 1360 His Val Tyr Gln Arg Ile Gln SerGlu Ile Pro Asn Glu Ala Pro Gln 1365 1370 1375 Glu Thr Pro Val Ala LeuGlu Val Thr Cys Tyr Val Asp Ser Glu Gly 1380 1385 1390 Asn Glu Val GlnGlu Thr Glu Glu Gly Thr His Asp Ala Pro Gly Ile 1395 1400 1405 Ile GlyAsp Lys Trp Gln Tyr Thr Gly Gln Thr Thr Thr Glu Asp Gly 1410 1415 1420Ile Thr Thr His Ile Tyr Gln Arg Ile Gln Ser Glu Ile Pro Asn Glu 14251430 1435 1440 Ala Pro Gln Glu Thr Pro Val Ala Leu Glu Val Thr Arg TyrVal Asp 1445 1450 1455 Ser Glu Gly Asn Glu Val Gln Glu Thr Glu Glu GlyThr His Gln Pro 1460 1465 1470 Pro Ser Ile Ile Gly Asp Lys Trp Gln TyrThr Gly Gln Thr Thr Thr 1475 1480 1485 Ala Asp Gly Ile Thr Thr Tyr ValTyr Glu Arg Ile Gln Ser Glu Ile 1490 1495 1500 Pro Asn Glu Ala Pro LysGlu Thr Pro Ile Gln Leu Glu Val Thr Arg 1505 1510 1515 1520 Tyr Val AspGly Glu Gly Asn Glu Val Gln Glu Thr Glu Glu Gly Thr 1525 1530 1535 HisHis Ala Pro Gly Ile Ile Gly Asp Lys Trp Gln Tyr Thr Gly Gln 1540 15451550 Thr Thr Thr Glu Ser Gly Ile Thr Thr His Val Tyr Glu Arg Ile Gln1555 1560 1565 Ser Glu Ile Pro Asn Glu Ala Pro Gln Glu Thr Pro Val GlnLeu Glu 1570 1575 1580 Val Thr Arg Tyr Val Asn Ser Glu Gly Asn Glu ValGln Glu Thr Glu 1585 1590 1595 1600 Glu Gly Thr His Gln Pro Pro Gly IleIle Gly Asp Lys Trp Gln Tyr 1605 1610 1615 Thr Gly Gln Thr Thr Thr AlaAsp Gly Ile Thr Thr Tyr Val Tyr Glu 1620 1625 1630 Arg Ile Gln Ser GluIle Pro Asn Glu Ala Pro Lys Glu Thr Pro Val 1635 1640 1645 Gln Leu GluVal Thr Arg Tyr Val Asp Thr Asp Gly Asn Glu Val Gln 1650 1655 1660 GluThr Glu Glu Gly Thr His Gln Pro Pro Gly Ile Ile Gly Asp Lys 1665 16701675 1680 Trp Gln Tyr Thr Gly Arg Val Thr Glu Lys Asp Gly Ile Thr ThrTyr 1685 1690 1695 Val Tyr Glu Arg Ile Gln Ser Ala Ile Pro Asn Glu AlaPro Gln Glu 1700 1705 1710 Thr Pro Val Gln Leu Glu Val Thr Arg Tyr ValAsp Ile Thr Gly Asn 1715 1720 1725 Glu Val Gln Glu Thr Glu Glu Gly ThrHis Gln Pro Arg Tyr Ile Ile 1730 1735 1740 Gly Asp Lys Trp Arg Tyr SerGly Val Thr Val Thr Glu Asn Gly Ile 1745 1750 1755 1760 Thr Lys His ValTyr Glu Arg Ile Gln Ser Lys Val Pro Asn Asp Ala 1765 1770 1775 Pro GlnGlu Thr Pro Val Gln Leu Glu Val Thr Arg Tyr Val Asp Pro 1780 1785 1790Glu Gly Asn Glu Ile Gln Glu Thr Thr Glu Gly Lys His Gln Pro Pro 17951800 1805 Gly Ile Ile Gly Asp Arg Trp Gln Tyr Thr Gly Lys Val Thr GluLys 1810 1815 1820 Asp Gly Ile Ile Thr Tyr Val Tyr Glu Arg Ile Gln SerGlu Ile Pro 1825 1830 1835 1840 Asn Asn Pro Pro Gln Glu Thr Pro Val GluLeu Glu Val Thr Arg Tyr 1845 1850 1855 Val Asp Gly Glu Gly Asn Glu ValGln Glu Thr Thr Glu Gly Lys His 1860 1865 1870 Gln Pro Pro Ser Ile IleGly Asp Arg Trp Gln Tyr Thr Gly Lys Val 1875 1880 1885 Thr Glu Lys AspGly Ile Thr Thr Tyr Val Tyr Glu Arg Ile Gln Ser 1890 1895 1900 Lys ValPro Asn Asp Ala Pro Arg Val Asp Ile Asp Glu Leu Lys Ile 1905 1910 19151920 Thr Ile Tyr Val Asp Thr Asn Gly Arg Glu Ile Val Pro Ser Arg Lys1925 1930 1935 Gly Gln Leu Pro Pro Glu Gln Phe Ile Gly Gln Asp Trp GlnTyr Thr 1940 1945 1950 Gly His Lys Ile Glu Lys Asp Gly Ile Thr Thr TyrIle Tyr Lys Lys 1955 1960 1965 Val Glu Asn Ala Val Pro Ala Lys Gln LeuLys Lys Thr Lys His Asn 1970 1975 1980 Thr Gln Ser Glu Ser Gln Phe LysHis Thr Pro Gln Val Lys Gln Gln 1985 1990 1995 2000 Leu Val Lys Tyr HisAsn Val Lys Glu Gln Arg Ser Ile Glu Lys Ser 2005 2010 2015 Glu His ThrAsp Met His Val Ser Glu Leu Pro Glu Thr Gly Glu Thr 2020 2025 2030 AlaAsn Lys Asn Gly Leu Ile Gly Gly Leu Leu Ile Ala Ile Gly Ala 2035 20402045 Phe Phe Val Thr Lys Arg Lys Lys Glu Asn Thr Lys 2050 2055 2060 31524 DNA Staphylococcus aureus 3 ttgaaaaata aattgctagt tttatcattgggagcattat gtgtatcaca aatttgggaa 60 agtaatcgtg cgagtgcagt ggtttctggggagaagaatc catatgtatc tgagtcgttg 120 aaactgacta ataataaaaa taaatctagaacagtagaag agtataagaa aagcttggat 180 gatttaatat ggtcctttcc aaacttagataatgaaagat ttgataatcc tgaatataaa 240 gaagctatga aaaaatatca acagagatttatggctgaag atgaggcttt gaagaaattt 300 tttagtgaag agaaaaaaat aaaaaatggaaatactgata atttagatta tctaggatta 360 tctcatgaaa gatatgaaag tgtatttaatactttgaaaa aacaaagtga ggagttctta 420 aaagaaattg aagatataaa aaaagataaccctgaattga aagactttaa tgaagaggag 480 caattaaagt gcgacttaga attaaacaaattagaaaatc agatattaat gttaggtaaa 540 acattttatc aaaactatag agatgatgttgaaagtttat atagtaagtt agatttaatt 600 atgggatata aagatgaaga aagagcaaataaaaaagcag ttaacaaaag gatgttagaa 660 aataaaaaag aagacttaga aaccataattgatgaatttt ttagtgatat agataaaaca 720 agacctaata atattcctgt tttagaagatgaaaaacaag aagagaaaaa tcataaaaat 780 atggctcaat taaaatctga cactgaagcagcaaaaagtg atgaatcaaa aagaagcaag 840 agaagtaaaa gaagtttaaa tactcaaaatcacaaacctg catctcaaga agtttctgaa 900 caacaaaaag ctgaatatga taaaagagcagaagaaagaa aagcgagatt tttggataat 960 caaaaaatta agaaaacacc tgtagtgtcattagaatatg attttgagca taaacaacgt 1020 attgacaacg aaaacgacaa gaaacttgtggtttctgcac caacaaagaa accaacatca 1080 ccgactacat atactgaaac aacgacacaggtaccaatgc ctacagttga gcgtcaaact 1140 cagcaacaaa ttatttataa tgcaccaaaacaattggctg gattaaatgg tgaaagtcat 1200 gatttcacaa caacgcatca atcaccaacaacttcaaatc acacgcataa taatgttgtt 1260 gaatttgaag aaacgtctgc tttacctggtagaaaatcag gatcactggt tggtataagt 1320 caaattgatt cttctcatct aactgaacgtgagaagcgtg taattaagcg tgaacacgtt 1380 agagaagctc aaaagttagt tgataattataaagatacac atagttataa agaccgaata 1440 aatgcacaac aaaaagtaaa tactttaagtgaaggtcatc aaaaacgttt taataaacaa 1500 atcaataaag tatataatgg caaa 1524 4517 PRT Staphylococcus aureus 4 Leu Gly Lys Ile Lys Glu Lys Ile Gln LeuLys Asn Lys Leu Leu Val 1 5 10 15 Leu Ser Leu Gly Ala Leu Cys Val SerGln Ile Trp Glu Ser Asn Arg 20 25 30 Ala Ser Ala Val Val Ser Gly Glu LysAsn Pro Tyr Val Ser Glu Ser 35 40 45 Leu Lys Leu Thr Asn Asn Lys Asn LysSer Arg Thr Val Glu Glu Tyr 50 55 60 Lys Lys Ser Leu Asp Asp Leu Ile TrpSer Phe Pro Asn Leu Asp Asn 65 70 75 80 Glu Arg Phe Asp Asn Pro Glu TyrLys Glu Ala Met Lys Lys Tyr Gln 85 90 95 Gln Arg Phe Met Ala Glu Asp GluAla Leu Lys Lys Phe Phe Ser Glu 100 105 110 Glu Lys Lys Ile Lys Asn GlyAsn Thr Asp Asn Leu Asp Tyr Leu Gly 115 120 125 Leu Ser His Glu Arg TyrGlu Ser Val Phe Asn Thr Leu Lys Lys Gln 130 135 140 Ser Glu Glu Phe LeuLys Glu Ile Glu Asp Ile Lys Lys Asp Asn Pro 145 150 155 160 Glu Leu LysAsp Phe Asn Glu Glu Glu Gln Leu Lys Cys Asp Leu Glu 165 170 175 Leu AsnLys Leu Glu Asn Gln Ile Leu Met Leu Gly Lys Thr Phe Tyr 180 185 190 GlnAsn Tyr Arg Asp Asp Val Glu Ser Leu Tyr Ser Lys Leu Asp Leu 195 200 205Ile Met Gly Tyr Lys Asp Glu Glu Arg Ala Asn Lys Lys Ala Val Asn 210 215220 Lys Arg Met Leu Glu Asn Lys Lys Glu Asp Leu Glu Thr Ile Ile Asp 225230 235 240 Glu Phe Phe Ser Asp Ile Asp Lys Thr Arg Pro Asn Asn Ile ProVal 245 250 255 Leu Glu Asp Glu Lys Gln Glu Glu Lys Asn His Lys Asn MetAla Gln 260 265 270 Leu Lys Ser Asp Thr Glu Ala Ala Lys Ser Asp Glu SerLys Arg Ser 275 280 285 Lys Arg Ser Lys Arg Ser Leu Asn Thr Gln Asn HisLys Pro Ala Ser 290 295 300 Gln Glu Val Ser Glu Gln Gln Lys Ala Glu TyrAsp Lys Arg Ala Glu 305 310 315 320 Glu Arg Lys Ala Arg Phe Leu Asp AsnGln Lys Ile Lys Lys Thr Pro 325 330 335 Val Val Ser Leu Glu Tyr Asp PheGlu His Lys Gln Arg Ile Asp Asn 340 345 350 Glu Asn Asp Lys Lys Leu ValVal Ser Ala Pro Thr Lys Lys Pro Thr 355 360 365 Ser Pro Thr Thr Tyr ThrGlu Thr Thr Thr Gln Val Pro Met Pro Thr 370 375 380 Val Glu Arg Gln ThrGln Gln Gln Ile Ile Tyr Asn Ala Pro Lys Gln 385 390 395 400 Leu Ala GlyLeu Asn Gly Glu Ser His Asp Phe Thr Thr Thr His Gln 405 410 415 Ser ProThr Thr Ser Asn His Thr His Asn Asn Val Val Glu Phe Glu 420 425 430 GluThr Ser Ala Leu Pro Gly Arg Lys Ser Gly Ser Leu Val Gly Ile 435 440 445Ser Gln Ile Asp Ser Ser His Leu Thr Glu Arg Glu Lys Arg Val Ile 450 455460 Lys Arg Glu His Val Arg Glu Ala Gln Lys Leu Val Asp Asn Tyr Lys 465470 475 480 Asp Thr His Ser Tyr Lys Asp Arg Ile Asn Ala Gln Gln Lys ValAsn 485 490 495 Thr Leu Ser Glu Gly His Gln Lys Arg Phe Asn Lys Gln IleAsn Lys 500 505 510 Val Tyr Asn Gly Lys 515 5 24 PRT Staphylococcuslugdunensis 5 Trp Gln Tyr Thr Gly Gln Thr Thr Thr Glu Asp Gly Ile ThrThr His 1 5 10 15 Ile Tyr Gln Arg Ile Gln Ser Glu 20 6 26 PRTStaphylococcus aureus 6 Thr Ser Pro Thr Thr Tyr Thr Glu Thr Thr Thr GlnVal Pro Met Pro 1 5 10 15 Thr Val Glu Arg Gln Thr Gln Gln Gln Ile 20 257 67 PRT Staphylococcus lugdunensis 7 Ile Pro Asn Asp Ala Pro Gln GluThr Pro Val Ala Leu Glu Val Thr 1 5 10 15 Arg Tyr Val Asp Ser Glu GlyAsn Glu Val Gln Glu Thr Glu Glu Gly 20 25 30 Thr His Asp Ala Pro Gly IleIle Ala Asp Lys Trp Gln Tyr Thr Gly 35 40 45 Gln Thr Ala Ala Glu Asn GlyIle Thr Thr His Val Tyr Gln Arg Ile 50 55 60 Gln Ser Glu 65 8 67 PRTStaphylococcus lugdunensis 8 Ile Pro Asn Asp Ala Pro Gln Glu Thr Pro ValAla Leu Glu Val Thr 1 5 10 15 Arg Tyr Val Asp Ser Glu Gly Asn Glu ValGln Glu Thr Glu Glu Gly 20 25 30 Thr His Asp Ala Pro Gly Ile Ile Ala AspLys Trp Gln Tyr Thr Gly 35 40 45 Gln Thr Ala Ala Glu Asn Gly Ile Thr ThrHis Val Tyr Gln Arg Ile 50 55 60 Gln Ser Glu 65 9 67 PRT Staphylococcuslugdunensis 9 Ile Pro Asn Asp Ala Pro Gln Glu Thr Pro Val Ala Leu GluVal Thr 1 5 10 15 Arg Tyr Val Asp Ser Glu Gly Asn Glu Val Gln Glu ThrGlu Glu Gly 20 25 30 Thr His Asp Ala Pro Gly Ile Ile Ala Asp Lys Trp GlnTyr Thr Gly 35 40 45 Gln Thr Ala Ala Glu Asn Gly Ile Thr Thr His Val TyrGln Arg Ile 50 55 60 Gln Ser Glu 65 10 67 PRT Staphylococcus lugdunensis10 Ile Pro Asn Glu Ala Pro Lys Glu Thr Pro Ile Gln Leu Glu Val Thr 1 510 15 Arg Tyr Val Asp Gly Glu Gly Asn Glu Val Gln Glu Thr Glu Glu Gly 2025 30 Thr His His Ala Pro Gly Ile Ile Gly Asp Lys Trp Gln Tyr Thr Gly 3540 45 Gln Thr Thr Thr Glu Ser Gly Ile Thr Thr His Val Tyr Glu Arg Ile 5055 60 Gln Ser Glu 65 11 67 PRT Staphylococcus lugdunensis 11 Ile Pro AsnGlu Ala Pro Gln Glu Thr Pro Val Gln Leu Glu Val Thr 1 5 10 15 Arg TyrVal Asn Ser Glu Gly Asn Glu Val Gln Glu Thr Glu Glu Gly 20 25 30 Thr HisGln Pro Pro Gly Ile Ile Gly Asp Lys Trp Gln Tyr Thr Gly 35 40 45 Gln ThrThr Thr Ala Asp Gly Ile Thr Thr Tyr Val Tyr Glu Arg Ile 50 55 60 Gln SerGlu 65 12 67 PRT Staphylococcus lugdunensis 12 Ile Pro Asn Glu Ala ProLys Glu Thr Pro Val Gln Leu Glu Val Thr 1 5 10 15 Arg Tyr Val Asp ThrAsp Gly Asn Glu Val Gln Glu Thr Glu Glu Gly 20 25 30 Thr His Gln Pro ProGly Ile Ile Gly Asp Lys Trp Gln Tyr Thr Gly 35 40 45 Arg Val Thr Glu LysAsp Gly Ile Thr Thr Tyr Val Tyr Glu Arg Ile 50 55 60 Gln Ser Ala 65 1367 PRT Staphylococcus lugdunensis 13 Ile Pro Asn Glu Ala Pro Gln Glu ThrPro Val Gln Leu Glu Val Thr 1 5 10 15 Arg Tyr Val Asp Ile Thr Gly AsnGlu Val Gln Glu Thr Glu Glu Gly 20 25 30 Thr His Gln Pro Arg Tyr Ile IleGly Asp Lys Trp Arg Tyr Ser Gly 35 40 45 Val Thr Val Thr Glu Asn Gly IleThr Lys His Val Tyr Glu Arg Ile 50 55 60 Gln Ser Lys 65 14 67 PRTStaphylococcus lugdunensis 14 Val Pro Asn Asp Ala Pro Gln Glu Thr ProVal Gln Leu Glu Val Thr 1 5 10 15 Arg Tyr Val Asp Pro Glu Gly Asn GluIle Gln Glu Thr Thr Glu Gly 20 25 30 Lys His Gln Pro Pro Gly Ile Ile GlyAsp Arg Trp Gln Tyr Thr Gly 35 40 45 Lys Val Thr Glu Lys Asp Gly Ile IleThr Tyr Val Tyr Glu Arg Ile 50 55 60 Gln Ser Glu 65 15 67 PRTStaphylococcus lugdunensis 15 Ile Pro Asn Asn Pro Pro Gln Glu Thr ProVal Glu Leu Glu Val Thr 1 5 10 15 Arg Tyr Val Asp Gly Glu Gly Asn GluVal Gln Glu Thr Thr Glu Gly 20 25 30 Lys His Gln Pro Pro Ser Ile Ile GlyAsp Arg Trp Gln Tyr Thr Gly 35 40 45 Lys Val Thr Glu Lys Asp Gly Ile ThrThr Tyr Val Tyr Glu Arg Ile 50 55 60 Gln Ser Lys 65 16 67 PRTStaphylococcus lugdunensis 16 Val Pro Asn Asp Ala Pro Arg Val Asp IleAsp Glu Leu Lys Ile Thr 1 5 10 15 Ile Tyr Val Asp Thr Asn Gly Arg GluIle Val Pro Ser Arg Lys Gly 20 25 30 Gln Leu Pro Pro Glu Gln Phe Ile GlyGln Asp Trp Gln Tyr Thr Gly 35 40 45 His Lys Ile Glu Lys Asp Gly Ile ThrThr Tyr Ile Tyr Lys Lys Val 50 55 60 Glu Asn Ala 65 17 10 PRTStaphylococcus aureus 17 Val Val Ser Gly Glu Lys Asn Pro Tyr Val 1 5 10

1. von Willebrand factor binding protein or polypeptide fromStaphylococci having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NOS: 5-17, andantigen determinant comprising parts thereof.
 2. Recombinant DNAmolecule comprising a nucleotide sequence coding for a protein orpolypeptide according to claim
 1. 3. Recombinant DNA molecule accordingto claim 2, comprising at least one nucleotide sequence selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 3, and nucleotidesequences coding for proteins and peptides having amino acid sequencesselected from SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NOS: 5-17, andantigen determinant comprising parts thereof.
 4. Plasmid, phage orphagemid comprising a DNA molecule according to claim 2 or
 3. 5.Microorganism comprising at least one recombinant DNA molecule accordingto claim 2 or 3, or at least one plasmid, phage or phagemid according toclaim
 4. 6. Method for producing a von Willebrand factor binding proteinor a polypeptide thereof, comprising the steps of introducing at leastone recombinant DNA molecule according to claim 2 or 3 in amicroorganism, culturing said microorganism in a suitable medium, andisolating the protein thus formed by chromatographic purification. 7.Method for producing a von Willebrand factor binding protein orpolypeptide thereof, comprising the step of expressing at least onerecombinant protein according to claim 1 on a phage particle to producea phage particle that shows von Willebrand factor binding activity. 8.Method of blocking the adherence of a Staphylococcus to surfaces,comprising addition of a protein according to claim 1, or an antibodyaccording to claim 11, to a medium containing said Staphylococcus. 9.Method according to claim 10, wherein the Staphylococcus is selectedfrom S. lugdunensis and S. aureus.
 10. Immobilized protein or peptideaccording to claim
 1. 11. Antibodies specifically binding to a proteinor peptide according to claim
 1. 12. Immunogen comprising a protein orpeptide according to claim 1 or
 10. 13. Method of purifying vonWillebrand factor from a complex solution comprising chromatography withthe immobilized protein of claim
 10. 14. Method of determining thepresence of von Willebrand factor in a complex solution comprising thestep of using a protein or peptide according to claim 1 or 10.